The Effectiveness of Biostimulation, Bioaugmentation and Sorption-Biological Treatment of Soil Contaminated with Petroleum Products in the Russian Subarctic

microorganisms

Article The Effectiveness of Biostimulation, Bioaugmentation and Sorption-Biological Treatment of Soil Contaminated with Petroleum Products in the Russian Subarctic

Vladimir A. Myazin 1,* , Maria V. Korneykova 1,2,*, Alexandra A. Chaporgina 1 , Nadezhda V. Fokina 1 and Galina K. Vasilyeva 3

1 Institute of North Industrial Ecology Problems—Subdivision of the Federal Research Centre “Kola Science Centre of Russian Academy of Science”, 184209 Apatity, Russia; [email protected] (A.A.C.); [email protected] (N.V.F.) 2 Agrarian Technological Institute, Рeoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russia 3 Institute of Physicochemical and Biological Problems in Soil Science Russian Academy of Science, 142290 Puschino, Russia; [email protected] * Correspondence: [email protected] (V.A.M.); [email protected] (M.V.K.)

Abstract: The effectiveness of different bioremediation methods (biostimulation, bioaugmentation, the sorption-biological method) for the restoration of soil contaminated with petroleum products in the Russian Subarctic has been studied. The object of the study includes soil contaminated for 20 years with petroleum products. By laboratory experiment, we established ﬁve types of microfungi Citation: Myazin, V.A.; Korneykova, that most intensively decompose petroleum hydrocarbons: Penicillium canescens st. 1, Penicillium M.V.; Chaporgina, A.A.; Fokina, N.V.; simplicissimum st. 1, Penicillum commune, Penicillium ochrochloron, and Penicillium restrictum. One day Vasilyeva, G.K. The Effectiveness of after the start of the experiment, 6 to 18% of the hydrocarbons decomposed: at 3 days, this was 16 Biostimulation, Bioaugmentation and Sorption-Biological Treatment of Soil to 49%; at 7 days, 40 to 73%; and at 10 days, 71 to 87%. Penicillium commune exhibited the greatest Contaminated with Petroleum degrading activity throughout the experiment. For soils of light granulometric composition with Products in the Russian Subarctic. a low content of organic matter, a more effective method of bioremediation is sorption-biological Microorganisms 2021, 9, 1722. treatment using peat or granulated activated carbon: the content of hydrocarbons decreased by https://doi.org/10.3390/ an average of 65%, which is 2.5 times more effective than without treatment. The sorbent not microorganisms9081722 only binds hydrocarbons and their toxic metabolites but is also a carrier for hydrocarbon-oxidizing microorganisms and prevents nutrient leaching from the soil. High efﬁciency was noted due to the Academic Editor: Persiani Anna biostimulation of the native hydrocarbon-oxidizing microfungi and bacteria by mineral fertilizers Maria and liming. An increase in the number of microfungi, bacteria and dehydrogenase activity indicate the presence of a certain microbial potential of the soil and the ability of the hydrocarbons to produce Received: 28 June 2021 biochemical oxidation. The use of the considered methods of bioremediation will improve the Accepted: 9 August 2021 Published: 13 August 2021 ecological state of the contaminated area and further the gradual restoration of biodiversity.

Keywords: petroleum-contaminated soil; bioremediation; sorbents; microfungi; mycoremediation; Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in hydrocarbon-oxidizing microorganisms; dehydrogenase activity; granular activated carbon; peat published maps and institutional afﬁl- iations.

1. Introduction Currently, because of anthropogenic activities, there is large-scale pollution of the Copyright: © 2021 by the authors. environment with toxic substances. Petroleum and petroleum products are major pollu- Licensee MDPI, Basel, Switzerland. tants among all contaminants entering the environment. Contamination with this type This article is an open access article of pollutant is also relevant for regions where petroleum production and reﬁning are distributed under the terms and not carried out. In this case, petroleum depots, objects of the fuel and energy complex, conditions of the Creative Commons and large industrial enterprises with motor transport divisions and gas stations in their Attribution (CC BY) license (https:// structure become potentially dangerous sources of soil pollution. Spills of petroleum or creativecommons.org/licenses/by/ petroleum products and the pollution of natural environments occur during their storage, 4.0/).

Microorganisms 2021, 9, 1722. https://doi.org/10.3390/microorganisms9081722 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 1722 2 of 18

transportation, shipment and bunkering, as well as resulting from the wearing down of equipment. The soil undergoes a special load—the degradation of its morphological and physicochemical properties, significant suppression of the soil’s microorganism activity and a decrease in its self-cleaning ability [1,2]. Contaminated soil ceases to perform the main ecosystem functions associated with its chemical, physicochemical, and biochemical properties. As a result of the degradation processes, the content and reserves of humus in the soil decrease; its qualitative compositional changes; the processes of nitrogen fixation, ammonification, nitrification, and mineralization are inhibited; the nutrient regime of the soil deteriorates; its enzymatic activity decreases; and the pH value and redox potential of the soil often change [2,3]. The need to develop effective methods of treatments and technologies for recovering petroleum-contaminated soils is one of the main environmental problems in the northern regions of Russia. In the Arctic zone of Russia, there is currently a problem with eliminating the consequences of accumulated environmental damage. There are areas contaminated with fuels and lubricants as a result of past economic activities where soil cleaning and recovery have not been carried out for many years [4,5]. Microbiological methods of environmental treatment from petroleum pollution are ecologically promising. The currently applied methods of cleaning oil-contaminated areas provide for the excavation of contaminated soil, its movement, and cleaning by physical, chemical or biological methods. Cleaned soils are used mainly in road construction. To restore the fertile layer in the cleaning area, natural soil is used. Remediation requires significant excavation and storage of soil, which will lead to an increase in the disturbed land area. All these manipulations change the natural soil profile, generate waste, and use additional soil resources [6]. These include biostimulation, which is based on the addition of extra sources of carbon and nutrients to stimulate microbial activity, and bioaugmentation—the introduction of cultures of petroleum-oxidizing microorganisms [3,7–9]. Recently, special attention has been paid to the possibility of using fungi for bioremediation (mycoremediation). Fungi are able to survive in adverse environmental conditions as well as to transform pollutants that are inaccessible or hardly accessible to soil bacteria [10–13]. There are a number of works demonstrating the successful application of mycoremediation [14–18]. The correct selection of fungi with a powerful degrading potential is the main criterion for the successful application of this technology. The use of the bioremediation method does not always give good results, especially in arctic and subarctic conditions. This is due to the low biological activity of the soils of the northern regions of Russia and the high vulnerability of the natural ecosystems. The solution to this problem could be the technology of sorption-biological soil treatment based on the use of various sorbents [19–21]. The use of sorbents accelerates the processes of bioremediation and land reclamation, thereby expanding the possibilities of biological treatment. Sorbents serve as a source of nitrogen and phosphorus and a place of localization of microorganisms-destructors, and they increase the moisture capacity and aeration of soils [22]. Studies carried out earlier in the central region of Russia made it possible to create an effective method of the sorption-biological treatment of soils contaminated with organic pollutants [23–27]. The use of granular activated carbon (GAC) can accelerate the processes of the bioremediation of soils contaminated with diesel fuel, used engine oil, and petroleum [28,29]. In laboratory conditions, the efficiency of the sorption-biological treatment of the contaminated soils of the Murmansk region has been shown. The introduction of optimal doses of sorbents into the soil make favorable conditions for the activation of indigenous or specially introduced microorganism-degraders [30]. The aim of this work is to determine the influence of bioremediation methods (biostimulation, bioaugmentation, sorption-biological treatment) on biological indicators and the content of hydrocarbons in contaminated soil. Microorganisms 2021, 9, 1722 3 of 18

2. Materials and Methods 2.1. The Territory of the Study The study was carried out in the area of Mount Kaskama, located in the north of the European part of Russia (Murmansk region (69◦16016.100 N; 29◦27037.900 E). This site belongs to the boreal–subarctic forest–tundra zone with a predominance of sparse pine forests (Pinus sylvestris lapponiсa)[31].

2.2. Climate This territory belongs to the Atlantic–Arctic region, which has two climatic zones: subarctic and temperate. The strong inﬂuence of the Atlantic has a softening effect on air temperature and increases humidity. The average air temperature in the coldest months (i.e., January, February) is −11 ◦C and in the warmest month (July) is + 12 ◦C. The average wind speed is 3.7 m/s and relative humidity is 68% [32]. The polar night is typical in winter and the polar day in summer.

2.3. Soils The undisturbed areas are dominated by folic albic podzol with slightly humiﬁed forest litter, a pH of 4.5–5.1, and a mineral proﬁle of up to 40–60 cm thick [33,34]. As a result of anthropogenic impact over a long time, a contaminated site about 0.1 hectares has formed. The surface of the contaminated site is devoid of an organogenic horizon (Figure1 ) and is represented by a sandy fraction with a large amount of stony inclusions (Figure2). The density of the soil is 1300 kg/m 3, and the content of organic carbon in the 0–7 cm layer is 4.47–5.43%.

2.4. Sorbents As sorbents, we used GAC of the GAU VSK brand and neutralized peat. The GAC with a granule size of 2–3 mm consists of 87–97% carbon, and the rest is represented by oxygen- and sulfur-containing high-molecular compounds, metals, etc. The speciﬁc surface area of activated carbon is 550–2400 m2/g, and the sorption capacity in relation to hydrocarbons is 200–980 g/kg [35]. The organic sorbent is milled peat with a low degree of decomposition (no more than 35%), the mass fraction of moisture is no more than 65%, the pHKCl is no less than 5.2, and the organic matter content is no less than 70%.

Figure 1. Contaminated area. Microorganisms 2021, 9, 1722 4 of 18

Figure 2. Soil proﬁle in the contaminated area.

2.5. Associations of Hydrocarbon Oxidizing Microorganisms In the field experiment, we used associations of hydrocarbon-oxidizing microorganisms (HOM), consisting of microfungi and bacteria that destroy petroleum products, isolated earlier from the contaminated soils of the Kola Peninsula. Previously, we investigated the petroleum-degrading activity of 81 fungi strains from 30 genera (Acremonium, Alternaria, Aspergillus, Aureobasidium, Botrytis, Cephalospo- rium, Cephalotrichum, Chaetomium, Cladosporium, Clonostachys, Fusicolla, Gibberella, Gibellulopsis, Gongronella, Humicola, Lecanicillium, Mucor, Penicillium, Pseudogym- noascus, Rhizopus, Rhodotorula, Scopulariopsis, Stachybotrys, Streptothrix, Talaromyces, Torula, Trichoderma, Ulocladium, Umbelopsis, Wallrothiella) in laboratory experiments. The cultivation of fungi was carried out in Erlenmeyer flasks with 50 mL of Czapek’s liquid nutrient medium of the following composition in g/L: NaNO3 at 3.0, KH2PO4 at 1.0, MgSO4 at 0.5, KCl at 0.5, and FeSO4 at 0.01. We inoculated 50 mL of the nutrient medium with 5 mL of a microfungi suspension with a density of 105–106 CFU in 1 mL. As a carbon source, oil from Prirazlomnoye field (Arctic Oil (ARCO) is distinguished by high density (about 910 kg/m3), medium sulfur content and low paraffin content) was introduced into the medium at a concentration of 1% by volume. The experiment was repeated 3 times. A sterile petroleum-free medium without fungi was used as a control. Incubation was carried out in a thermostat at 27 ◦C for 14 days. The biomass of microfungi was determined by drying it at 105 ◦C to absolutely dry weight. The petroleum degradation by the most active strains of microscopic fungi isolated of the previous experiment (i.e., Penicillium canescens st.1, Penicillium commune, Penicillium ochrochloron, Penicillium restrictum, Penicillium simplicissimum st.1) was studied in a time gradient: after 1, 3, 7, and 10 days. At the final stage, the degrading activity of fungi associations and bacteria was investigated. Active strains of fungi (P. commune, P. simplicissimum st. 1, Penicillium canescens st. 1) and hydrocarbon-oxidizing bacteria (Microbacterium paraoxydans, Pseudomonas fluorescens, Pseudomonas baetica, Pseudomonas putida), previously isolated from the soils of the Kola Peninsula. were used in laboratory experiment [36,37]. Bacterial biomass was produced in meat-peptone broth in a laboratory fermenter (BIOSTAT® A plus, Sartorius, Germany) at a temperature 27 ◦C and aeration for 3 days. The density of the bacterial suspension was 108–109 cells/mL. Cell density was determined by fluorescence microscopy using polycarbonate membrane filters. Regions of the 16S and Internal Transcribed Spacer (ITS) gene obtained from these bacterial strains were deposited in the NCBI GenBank database under the accession numbers KM 288708, KM 288709, and KM 216318. Microscopic fungi were produced in the same way as in the previous experiment. To prepare an association of hydrocarbon-oxidizing microorganisms, bacterial and fungal suspensions were mixed in equal proportions in meat-peptone broth. Microorganisms 2021, 9, 1722 5 of 18

2.6. Experiment Design Experimental plots with a size of 1 m2 in triplicate were laid at the western foot of the mountain. The design of the ﬁeld experiment is presented in Table1.

Table 1. The experiment design.

HOM Activated N P K , Dolomite 16 16 16 Association Variant Carbon (g N, P O , Flour, Peat, L/m2 2 5 Suspension, (GAC), g/m2 K O/m2) g CaCO /m2 2 3 L/m2 K----- NPK - 25 200 - - BP - 25 200 2 - GAC1 1300 25 200 - - GAC3 3900 25 200 - - Peat - 25 200 - 16

To activate aboriginal petroleum-degrading microorganisms (biostimulation) and remove nutritional deficiencies, complex mineral fertilizer (16% N, 16% P2O5, and 16% K2O) and dolomite flour (CaCO3 content of 80%) were added to all plots except for the control (K). The amount of fertilizer was calculated based on the content of hydrocarbons in the soil (ratio C:N = 100:1), and the amount of dolomite flour was calculated based on the pH value. After the addition of fertilizer and dolomite flour, an association of HOM (BP) in the amount of 2 L/m2, GAC in the amount of 1 and 3 wt. % (GAC1 and GAC3, respectively), and peat (Peat) in an amount of 16 l/m2 were added to various sites. The one variant (NPK) with fertilizer and dolomite flour was left without adding sorbents and HOM. The top layer of the soil in all variants was mixed to a depth of 7–10 cm. After 12 months (at the beginning of the next growing season), the soil amendments were repeated. Soil samples were taken from each experimental site from a layer of 0–7 cm. The experiment was started in June 2019, the observation period was 15 months. Samples were taken during the growing season (0 and 12 months) and at the end of the growing season (3 and 15 months).

2.7. Determination of the Total Petroleum Hydrocarbons Content in Soil The content of total petroleum hydrocarbons (TPH) in the soil was determined by infrared spectrometry by using the AN-2 analyzer [38]. This method is based on the extraction of TPH from the soil with tetrachlorocarbon, the separation of oil products from the polar hydrocarbons in the column filled with aluminum oxide, and the further spectrophotometric identification of hydrocarbon content according to the absorption intensity of infrared radiation at fixed wavelengths 540 nm (Analyzer of oil products AN-2 “Neftekhimavtomatika”).

2.8. Determination of Physical-Chemical Properties of Soil The actual acidity of the soil was determined in water soil extracts in a ratio of 1:5 by the potentiometric method on a laboratory instrument, a Radelkis OP-300 pH meter with a combined pH electrode [39]. The instrument was calibrated in the pH range from 4.01 to 6.86 using buffer solutions prepared from standard titers. Soil humidity was measured by drying soil samples at 105 ◦C for at least 6 h to constant weight. Soil temperature was determined in the ﬁeld at a depth of 5 cm using a Hanna HI 145 electronic thermometer. Microorganisms 2021, 9, 1722 6 of 18

2.9. Biological Parameters For microbiological analysis on each experimental plot, soil samples were taken from a layer of 0–10 cm. The number of cultivated microscopic fungi was determined by the submerged inoculation method on a nutrient medium with the addition of lactic acid (4 mL/L) for bacteriostatic action. The number of cultivated bacteria was determined by the surface inoculation method on a nutrient medium for hydrocarbon-oxidizing bacteria with the addition of petroleum. The activity of soil dehydrogenase was determined by the colorimetric method based on the reduction of the colorless salt of 2,3,5-triphenyltetrazolium chloride to red triphenyl- formazan [40,41].

2.10. Statistical Processing Statistical analysis of the data was carried out using the applied programs Statistica 6.0 and Microsoft Excel 2007. Student's t-test was used to determine the reliability of the coefficients. Pearson's method (significance level 0.05) was used to calculate the correlation coefficient (r).

3. Results 3.1. Degradation Activity of Microfungi in Laboratory Experiments Based on the results obtained, a scale of degrading activity of microfungi to petroleum hydrocarbons has been developed (Table2). All species were categorized into three groups: (a) Species with high hydrocarbon oxidizing activity, reducing the petroleum hydrocarbon content by 80–98%. (b) Species with medium hydrocarbon oxidizing activity, reducing the petroleum hydrocarbon content by 50–79%. (c) Species with low hydrocarbon oxidizing activity, reducing the petroleum hydrocarbon content by 49% or less.

Table 2. Hydrocarbon oxidizing activity of fungi species for 14 days (the initial concentration of petroleum products is 1% by volume in the medium).

Petroleum Degradation, % of Degradation Activity, g Species of Microfungi Dry Biomass, g the Initial Amount Petroleum /g Microfungi group I—high activity Penicillium commune Thom 98 0.414 0.596 P. canescens (st.1) Sopp 98 0.372 0.867 P. simplicissimum (st.1) (Oudem.) Thom 96 0.353 0.687 P. canescens (st.2) Sopp 95 0.335 0.758 P. restrictum J.C. Gilman & E.V. Abbott 95 0.330 0.738 P. ochrochloron Biourge 94 0.337 0.615 P. velutinum J.F.H. Beyma 93 0.253 0.335 Ulocladium consortiale 92 0.343 0.518 (Thum.)E.G.Simmons P. implicatum Biourge 92 0.327 0.530 P. decumbens Thom 92 0.266 0.810 P. canescens (st.3) Sopp 88 0.229 0.651 P. simplicissimum (st.2) (Oudem.) Thom 87 0.311 0.519 P. miczynskii (st.1) K.M. Zaleski 85 0.294 0.573 Microorganisms 2021, 9, 1722 7 of 18

Table 2. Cont.

Petroleum Degradation, % of Degradation Activity, g Species of Microfungi Dry Biomass, g the Initial Amount Petroleum /g Microfungi P. spinulosum Thom. 84 0.245 0.847 P. jensenii (st.1) K.M. Zaleski 84 0.312 0.667 Fusarium solani (Mart.) Sacc. 84 0.310 0.251 Alternaria alternata (Fr.) Keissl. 83 0.304 0.515 F. oxysporum (st.1) Schltdl. 80 0.311 0.268 group II—medium activity P. aurantiogriseum (st.1) Dierckx 79 0.269 0.620 Rhizopus stolonifer (Ehrenb.) Vuill. 76 0.285 0.485 P. adametzii K.M. Zaleski 75 0.267 0.716 P. glabrum (Wehmer) Westling 74 0.248 0.406 P. spinulosum (st.2) Thom 74 0.281 0.463 P. miczynskii (st.2) K.M. Zaleski 73 0.265 0.525 Lecanicillium lecanii (Zimm.) Zare & 72 0.298 0.352 W. Gams Stachybotrys echinata (Rivolta) G. Sm., 70 0.239 0.406 P. canescens (st.4) Sopp. 70 0.295 0.466 Trichoderma viride Pers. 67 0.233 0.283 P. aurantiogriseum (st.2) Dierckx 67 0.247 0.418 P. nalgiovense Laxa 66 0.266 0.628 Umbelopsis isabellina (Oudem.) W. Gams 63 0.294 0.415 Cephalotrichum stemonitis (Pers.) Nees 62 0.257 0.563 Talaromyces stipitatus C.R. Benj. 62 0.270 0.361 Chaetomium bostrychodes Zopf 60 0.294 0.365 Acremonium egyptiacum (J.F.H. Beyma) 57 0.215 0.391 W. Gams, Fusicolla merismoides (Corda) Gräfenhan, 57 0.218 0.224 Seifert & Schroers Wallrothiella subiculosa Höhn. 55 0.241 0.300 P. multicolor Grig.-Man. & Porad. 50 0.231 0.407 group III—low activity L. psalliotae (Treschew) Zare & W. Gams 46 0.233 0.369 Clonostachys rosea (Link) Schroers, Samuels, Seifert & W. Gams 44 0.281 0.402 Umbelopsis longicollis (Dixon-Stew.) Y.N Wang, X.Y. Liu & R.Y. Zheng 41 0.230 0.391 Cephalosporium bonordenii Sacc. 37 0.204 0.191 Pseudogymnoascus pannorum (Link) Minnis & D.L. Lindner 33 0.233 0.185 Scopulariopsis communis (st.1) Bainier 33 0.195 0.168 P. thomii Maire 31 0.196 0.444 Microorganisms 2021, 9, 1722 8 of 18

Table 2. Cont.

Petroleum Degradation, % of Degradation Activity, g Species of Microfungi Dry Biomass, g the Initial Amount Petroleum /g Microfungi Aspergillus fumigatus Fresen. 30 0.287 0.438 P. jensenii K.M. Zaleski 30 0.241 0.332 Tr. koningii Oudem. 29 0.162 0.116 P. melinii Thom 29 0.274 0.419 P. aurantiogriseum (st.3) Dierckx 28 0.245 0.461 Phoma eupyrena Sacc. 28 0.236 0.513 Torula herbarum (Pers.) Link 27 0.180 0.186 Mucor hiemalis Wehmer 25 0.149 0.104 Ph. herbarum Westend. 25 0.205 0.236 Acr. charticola (Lindau) W. Gams 24 0.213 0.234 Tr. polysporum (Link) Rifai 23 0.136 0.044 Amorphotheca resinae Parbery 20 0.170 0.174 Gibberella fujikuroi (Sawada) Wollenw. 20 0.170 0.293 Acr. rutilum W. Gams 20 0.117 0.123 Tr. aureoviride Rifai 19 0.162 0.165 Clad. cladosporioides (Fresen.) G.A. de Vries 19 0.160 0.071 Gibellulopsis nigrescens (Pethybr.) Zare, W. Gams & Summerb. 17 0.222 0.183 P.aurantiogriseum(st.4) Dierckx 17 0.122 0.327 Aureobasidium microstictum (Bubák) W.B. Cooke 17 0.110 0.095 Sc. communis (st.2) Bainier 15 0.109 0.165 Gongronella butleri (Lendn.) Peyronel & Dal Vesco 13 0.107 0.109 Ph. glomerata (Corda) Wollenw. & Hochapfel 13 0.029 0.279 M.circinelloides Tiegh. 10 0.116 0.163 Rodotorula sp. 10 0.087 0.128 Aur. pullulans (de Bary & Löwenthal) G. Arnaud 9 0.054 0.097 Cph. nanum (Ehrenb.)S.Hughes 9 0.050 0.090 P. corylophilum Dierckx 9 0.091 0.501 Streptothrix luteola Foul. & P.C. Jones, 8 0.107 0.311 Tl. ﬂavus (Klocker) Stolk et Samson 8 0.126 0.289 P. raistrickii G. Sm. 7 0.084 0.423 Asp. repens (Corda) Sacc. 6 0.074 0.100 T. allii (Harz) Sacc. 5 0.083 0.141 Botrytis cinerea Pers. 5 0.065 0.164 F. oxisporum Schltdl. 4 0.053 0.178 P. spinulosum Thom 4 0.033 0.119 Humicola grisea Traaen 3 0.020 0.280 Microorganisms 2021, 9, 1722 9 of 18

The group of species with low hydrocarbon oxidizing activity, represented by 43 species, was the most diverse in terms of species composition. The fungi had a weak ability to grow on petroleum hydrocarbons and were not of particular interest for further study. The group of species with medium hydrocarbon oxidizing activity was represented by 20 species of microfungi. The group of species with high hydrocarbon oxidizing activity includes 18 strains of microfungi: Alternaria alternata, Fusarium oxysporum, F. solani, Penicillium canescens st. 1, st. 2, st. 3, P. commune, P. decumbens, P. implicatum, P. jensenii st. 1, P. miczynskii st. 1, P. nigricans, P. ochrochloron, P. restrictum, P. simplicissimum st. 1, st. 2, P. velutinum, and Ulocladium consortiale. Among these, in terms of species diversity, the genus Penicillium prevailed, the representatives of which are also resistant to other types of pollution [42,43]. The most intensive petroleum hydrocarbons decomposed ﬁve species of microfungi: Penicillium canescens st. 1, P. simplicissimum st. 1, P. commune, P. ochrochloron, and P. restrictum. One day after the start of the experiment, 6 to 18% of hydrocarbons decomposed. This grew in 3 days to 16 to 49%, in 7 days to 40 to 73%, and in 10 days to 71 to 87%. The Penicillium commune exhibited the greatest degrading activity throughout the experiment. In the experiment with associations of microorganisms, on the third day, there was a decrease in petroleum hydrocarbon content of 11 to 38%. On the seventh day, it was 17 to 83%, and on the tenth day, it was up to 94%. The associations of microfungi with bacteria were the most effective. It showed the best result on the third day of the experiment. The association of bacteria in this experiment was more passive and decreased only 20% of the petroleum hydrocarbon in 10 days. The most active association of microorganisms was used in a ﬁeld experiment to assess the effectiveness of the methods for the bioremediation of contaminated soils.

3.2. Analysis of the Bioremediation Effectiveness in a Field Experiment 3.2.1. Content of Total Petroleum Hydrocarbons The initial content of TPH in the contaminated soil at different sites ranged from 9385 to 46,143 mg/kg (Table3).

Table 3. The initial content of TPH and organic matter, humidity, pH and temperature of soil in the contaminated area.

TPH, mg/kg Organic Matter, % pH Humidity, % Temperature, ◦C 26,548 ± 2299 5.04 ± 0.29 5.93 ± 0.03 10.2 ± 3.1 21.5 ± 1.1

The TPH content in the soil in variant A decreased by 30% over 15 months due to the optimization of the air regime as a result of the periodic mixing of the upper layer. The application of mineral fertilizers decreased the concentration of hydrocarbons in the contaminated soil by an average of 47%. The use of the HOM association did not give additional advantages and reduced the TPH content by 45% from the initial value. The efficiency of the sorption-biological method of purification with the addition of GAC was 49–53%. The maximum efficiency was observed when we used peat: the TPH content decreased by an average of 65% (Figure3a). The content of extractable polar organic compounds (not hydrocarbons) decreases by 65–72% when using various methods of bioremediation and by 54% without bioremediation (Figure3b). Microorganisms 2021, 9, x FOR PEER REVIEW 9 of 17

Table 3. The initial content of TPH and organic matter, humidity, pH and temperature of soil in the contaminated area.

Organic Matter, TPH, mg/kg рН Humidity, % Temperature, °С % 26548 ± 2299 5.04 ± 0.29 5.93 ± 0.03 10.2 ± 3.1 21.5 ± 1.1

The TPH content in the soil in variant A decreased by 30% over 15 months due to the optimization of the air regime as a result of the periodic mixing of the upper layer. The application of mineral fertilizers decreased the concentration of hydrocarbons in the contaminated soil by an average of 47%. The use of the HOM association did not give additional advantages and reduced the TPH content by 45% from the initial value. The efficiency of the sorption-biological method of purification with the addition of GAC was 49–53%. The maximum efficiency was observed when we used peat: the TPH content decreased by an average of 65% (Figure 3a). Microorganisms 2021, 9, 1722 The content of extractable polar organic compounds (not hydrocarbons) decreases by10 of 18 65–72% when using various methods of bioremediation and by 54% without bioremediation (Figure 3b).

FigureFigure 3. The 3. dynamicThe dynamic of the of TPH the content TPH content (a) and (a )high-molecular and high-molecular polar polarorganic organic compounds compounds (b) in (theb) in contaminated the contaminated soil soil with differentwith different methods methods of bioremediation. of bioremediation.

The minimumThe minimum rate of rate hydrocarbon of hydrocarbon decay decay was at was the at variants the variants K and K BP and when BP when we used we used the HOMthe HOM association: association: 24 and 24 28 and mg 28 TPH/kg mg TPH/kg per day, per respectively. day, respectively. With Withthe sorption- the sorption- biologicalbiological method method of purification of purification and using and GAC, using the GAC, rate the of hydrocarbons rate of hydrocarbons decay was decay 42– was 45, and42–45, with and the addition with the of addition peat, it ofwas peat, 38 mg it was TPH/kg 38 mg per TPH/kg day. The per average day. Therate averageof TPH rate decayof when TPH using decay the when biostimulation using the biostimulation method was 47 method mg TPH/kg was47 per mg day. TPH/kg The maximum per day. The rate ofmaximum TPH decay rate in ofall TPHvariants decay of the in allfield variants experiment of the was field observed experiment in the was first observed 3 months in the and reachedfirst 3 months 97–105 and mg reachedTPH/kg 97–105per day mg when TPH/kg using perGAC. day when using GAC. The Thecontent content of high-molecular of high-molecular polar polar or organicganic compounds compounds also decreaseddecreased mostmost inten- intensivelysively inin thethe first first 3 months.3 months. In In both both cases, cases, in variants in variants NPK, NPK, BP, and BP, GAC1, and GAC1, we observed we a observeddecrease a decrease in the in rate the of rate decay of decay in the in winter the winter (0–9 mg/kg(0–9 mg/kg per day),per day), whereas whereas in the in variantsthe variantsGAC3 GAC3 and and Peat, Peat, the the rate rate of decay of decay reached reached 25 mg/kg 25 mg/kg per per day. day. The sorption-biologicalThe sorption-biological method method was more was moreeffective effective than thanthe other the other methods methods according according to theto results the results of the of first the growing first growing season. season. An undoubted An undoubted advantage advantage of this of method this method is the is the ability of the microorganisms on the sorbent to oxidize the hydrocarbons and organic compounds of other classes even in winter. Therefore, by the beginning of the second growing season, the content of the hydrocarbons in these areas decreased more.

The data from the analysis of the hydrocarbon content in the soil were used to calculate the time of their almost complete decomposition, i.e., by 99% (T99), according to the exponential dependence [44]. The studies showed that the decomposition of hydrocarbons was accelerated by 1.7 and 2.9 times with bioremediation (Table4). When using peat, almost complete soil cleaning will occur in 5.4 years, without bioremediation—in 16.1 years. Microorganisms 2021, 9, x FOR PEER REVIEW 10 of 17

ability of the microorganisms on the sorbent to oxidize the hydrocarbons and organic compounds of other classes even in winter. Therefore, by the beginning of the second growing season, the content of the hydrocarbons in these areas decreased more. The data from the analysis of the hydrocarbon content in the soil were used to calculate the time of their almost complete decomposition, i.e., by 99% (T99), according to Microorganisms 2021, 9, 1722 11 of 18 the exponential dependence [44]. The studies showed that the decomposition of hydrocarbons was accelerated by 1.7 and 2.9 times with bioremediation (Table 4). When using peat, almost complete soil Tablecleaning 4. Time willof occur practically in 5.4 fullyears, degradation without bioremediation (Т99) and degradation—in 16.1 to approximateyears. permissible concentration (ТAPC, 700 mg/kg) of petroleum hydrocarbons in soil. Table 4. Time of practically full degradation (Т99) and degradation to approximate permissible concentration (ТAPC, 700 mg/kg)The Rate of petroleum Constant hydrocarbons in soil. Variant of Hydrocarbons TAPC (Days) T99 (Days) The Rate ConstantDecomposition of Hydrocarbons Variant ТAPC (Days) Т99 (Days) KDecomposition 0.00079 4892 5816 K NPK 0.001400.00079 2699 4892 32985816 NPK 0.00140 2699 3298 BP 0.00135 2428 3420 BP 0.00135 2428 3420 GAC1 0.00168 2104 2746 GAC1 0.00168 2104 2746 GAC3GAC3 0.001510.00151 2501 2501 30533053 Peat Peat 0.002350.00235 1479 1479 19601960

3.2.2.3.2.2. SoilSoil HumidityHumidity andand TemperatureTemperature TheThe bioremediation bioremediation did did not not signiﬁcantly significantly affect affect the the soil soil temperature temperature measured measured at at the the timetime of of sampling sampling in in June June and and September.September. However, However, the the temperature temperature measured measured a a year year after after thethe startstart ofof thethe studystudy showedshowed thatthat onon thethe plotsplots withwith thethe peat,peat, therethere waswas a a tendency tendency to to decreasedecrease it. it. TheThe bioremediationbioremediation had had a a positive positive effect effect on on soil soil humidity. humidity. The The increase increase in in soil soil humidityhumidity could could be be facilitated facilitated by by the the biodegradation biodegradation of of hydrocarbons, hydrocarbons, which which could could lead lead to to thethe formation formation of of water. water. The The maximum maximum moisture moisture content content was was typical typical for for the the variant variant with with peat.peat. AnAn increaseincrease inin humidityhumidity cancan explainexplain thethe observedobserved tendencytendency towardtoward aa decrease decrease in in temperaturetemperature inin variantsvariants withwith peat.peat. InIn thethe autumn,autumn, thethe increaseincrease inin soilsoil humidity humidity onon the the reclaimedreclaimed sitessites waswas moremore pronouncedpronounced thanthan inin thethe summer, summer, when when the the moisture moisture content content droppeddropped to to a a minimum minimum (Figure (Figure4 ).4).

FigureFigure 4. 4.The The change change of of the the soil soil humidity humidity due due to to the the bioremediation. bioremediation.

3.2.3. Actual Acidity The acidity of the soil is one of the most important indicators of its condition and affects the vital activity of soil microorganisms as well as the speed and direction of the chemical and biochemical processes. Soil pH inﬂuences biodegradation through its effect on microbial activity, microbial community and diversity, enzymes that aid in the degradation processes. The optimum pH for biodegradation is alkaline or slightly acid [45]. pH values between 6.5 and 8.0 are considered optimum for oil degradation [46]. Microorganisms 2021, 9, x FOR PEER REVIEW 11 of 17

3.2.3. Actual Acidity The acidity of the soil is one of the most important indicators of its condition and affects the vital activity of soil microorganisms as well as the speed and direction of the

Microorganisms 2021, 9, 1722 chemical and biochemical processes. Soil pH influences biodegradation through its12 effect of 18 on microbial activity, microbial community and diversity, enzymes that aid in the degradation processes. The optimum pH for biodegradation is alkaline or slightly acid [45]. pH values between 6.5 and 8.0 are considered optimum for oil degradation [46]. Liming,Liming, thethe useuse ofof GACGAC andand peatpeat inin thethe processprocess ofof cleaningcleaning contaminatedcontaminated areas,areas, increasedincreased the the soil soil pH pH by by 0.3–0.8 0.3–0.8 units units (Figure (Figure5). 5). At At the the same same time, time, when when the upperthe upper layer layer of theof contaminatedthe contaminated soil issoil only is loosened, only loosened, soil acidiﬁcation soil acidification is observed, is observed, which can which adversely can affectadversely the state affect of the the state soil microbial of the soil community microbial community and is unfavorable and is unfavorable for plants. for plants. TheThe change change of of soil soil pH pH is is not not onlyonly aa consequenceconsequence of of liming liming but but also also the the buffering buffering effect effect ofof some some elements elements (potassium, (potassium, calcium, calcium, magnesium magnesium cations, cations, etc.) etc.) contained contained in in the the activated activated carbons,carbons, whichwhich waswas previously previously observed observed in in laboratory laboratory experiments experiments [ 29[29,30,47].,30,47]. Thus, Thus, the the useuse of of GAC GAC reduced reduced the negativethe negative consequences consequenc of applyinges of applying high doses high of mineraldoses of fertilizers mineral duringfertilizers bioremediation during bioremediation and maintained and maintained the soil pH the in soil the pH optimal in the rangeoptimal for range most for plants most andplants microorganisms. and microorganisms.

FigureFigure 5. 5.The The change change of of soil soil pH pH with with various various methods methods of of bioremediation. bioremediation.

3.2.4.3.2.4. TheThe NumberNumber ofof Hydrocarbon-OxidizingHydrocarbon-oxidizing MicroorganismsMicroorganisms TheThe numbernumber of hydrocarbon-oxidizing hydrocarbon-oxidizing microfungi microfungi in in the the soil soil varied varied from from 0.15 0.15 to 0.52 to 0.52thousand. thousand. CFU/g CFU/g (Figure (Figure 6a).6a). As As a aresult result of biostimulationbiostimulation andand sorption-biological sorption-biological treatmenttreatment of of the the site, site, an an increase increase in in the the number number of of microfungi microfungi was was already already noted noted (variants (variants NPK,NPK, GAC3,GAC3, andand Peat)Peat) inin the the ﬁrst first months. months. AfterAfter 1515 months,months, thethe maximum maximum numbernumber ofof microfungimicrofungi waswas observedobserved inin variantvariant BP,BP, which which is is due due to to the the repeated repeated introduction introduction of of the the associationassociation of of HOM HOM and and variant variant Peat Peat (7.82 (7.82 and and 12.13 12.13 thousand thousand CFU/g, CFU/g, respectively). respectively). The The highhigh number number of of microfungi microfungi with with variant variant Peat Peat is is due due to to the themicrobial microbial pool pool of of the thepeat peatitself, itself, which,which, accordingaccording toto the results obtained, obtained, was was more more effective effective than than the the microbial microbial pool pool of the of the HOM association. In variant K, the number of microfungi did not change during the HOM association. In variant K, the number of microfungi did not change during the experiment. This may be the result of a less favorable habitat for microfungi (low organic experiment. This may be the result of a less favorable habitat for microfungi (low organic matter content, very low humidity in the summer, and the high temperature and pH values matter content, very low humidity in the summer, and the high temperature and pH of the substrate). Further, it was noted that the number of microfungi increases in the values of the substrate). Further, it was noted that the number of microfungi increases in autumn under more favorable living conditions. the autumn under more favorable living conditions. Observations of the bacterial component of soil samples during the second growing season showed that the maximum number of bacteria in the soil of the recultivated areas was 6.5 million cells/g. Noteworthy is the low number of bacteria in variants NPK and BP at the beginning of the second growing season (Figure6b), which is probably due to the very low moisture content of these samples. An increase in the bacteria number in the variants GAC1, GAC3, and Peat is associated with an improvement of the physicochemical soil properties and the appearance of an additional source of organic carbon. Throughout the growing season, no signiﬁcant differences were found related to the number of bacteria in the soil and the addition of GAC and peat. Microorganisms 2021, 9, x FOR PEER REVIEW 12 of 17

Observations of the bacterial component of soil samples during the second growing season showed that the maximum number of bacteria in the soil of the recultivated areas was 6.5 million cells/g. Noteworthy is the low number of bacteria in variants NPK and BP at the beginning of the second growing season (Figure 6b), which is probably due to the very low moisture content of these samples. An increase in the bacteria number in the variants GAC1, GAC3, and Peat is associated with an improvement of the Microorganisms 2021, 9, 1722 physicochemical soil properties and the appearance of an additional source of organic13 of 18 carbon. Throughout the growing season, no significant differences were found related to the number of bacteria in the soil and the addition of GAC and peat.

FigureFigure 6.6. TheThe dynamicdynamic ofof thethe numbernumber ofof hydrocarbon-oxidizinghydrocarbon-oxidizing microfungimicrofungi (a)(a) andand bacteriabacteria (b)(b) inin soilsoil withwith differentdifferent methodsmethods ofof bioremediation.bioremediation.

InIn allall thethe samplessamples ofof contaminatedcontaminated soils,soils, aa veryvery lowlow speciesspecies diversitydiversity ofof bacteriabacteria waswas noted.noted. BacteriaBacteria ofof thethe genusgenus PseudomonasPseudomonas dominated;dominated; pigmentedpigmented formsforms ofof bacteriabacteria moremore resistantresistant toto pollutionpollution werewere presented.presented. ActinomycetesActinomycetes werewere foundfound inin smallsmall numbers.numbers. TheThe consideredconsidered bioremediationbioremediation methodsmethods ofof thethe petroleum-contaminatedpetroleum-contaminated areaarea hadhad aa positivepositive effecteffect onon thethe bacteriabacteria number.number. Sorption-biologicalSorption-biological methodsmethods ofof bioremediationbioremediation makemake itit possiblepossible toto increaseincrease thethe resistanceresistance ofof bacterialbacterial communitycommunity toto unfavorableunfavorable factors,factors, whichwhich isis conﬁrmedconfirmed byby aa higherhigher bacteriabacteria numbernumber atat thethe beginningbeginning ofofthe thesecond secondgrowing growing season. season.

3.2.5.3.2.5. SoilSoil DehydrogenaseDehydrogenase ActivityActivity Microorganisms are the main source of enzymes entering the soil. Changes in their Microorganisms are the main source of enzymes entering the soil. Changes in their composition and abundance in contaminated soil also affect the activity of soil enzymes. composition and abundance in contaminated soil also affect the activity of soil enzymes. Due to extracellular enzymatic activity, the soil metabolism can remain unchanged even in Due to extracellular enzymatic activity, the soil metabolism can remain unchanged even unfavorable conditions for the vital activity of microorganisms [48]. The most important in unfavorable conditions for the vital activity of microorganisms [48]. The most and widespread oil destructor enzymes of soil microorganisms are catalase and dehydro- important and widespread oil destructor enzymes of soil microorganisms are catalase and genase. Dehydrogenase is an intracellular enzyme, and therefore, its activity reﬂects the dehydrogenase. Dehydrogenase is an intracellular enzyme, and therefore, its activity functional state of the soil microbial community [49,50]. reflects the functional state of the soil microbial community [49,50]. The dehydrogenase activity in the contaminated soil before the bioremediation was The dehydrogenase activity in the contaminated soil before the bioremediation was 0.06 mg TPF/10 g, which corresponds to very weak biological activity. This may indicate 0.06 mg TPF/10 g, which corresponds to very weak biological activity. This may indicate the inhibition of the enzymatic dehydrogenation of hydrocarbons. The maximum values ofthe the inhibition enzyme of activity the enzymatic were observed dehydrogen at theation end ofof thehydrocarbons ﬁrst and second. The maximum growing seasons values inof thethe variantsenzyme activity NPK (0.18 were mg observed TPF/10 at g), th GAC3e end (0.21of the mg first TPF/10 and second g), and growing Peat (0.29 seasons mg TPF/10 g; Figure7), which, however, corresponds to weak enzymatic activity. More high values of enzymatic activity at the beginning of the second growing season in variants GAC3 and Peat correlate with the rate of the degradation of the hydrocarbons in these variants in winter. By the end of the second growing season, the enzyme activity in all the variants, except for the variant BP, slightly decreased, despite the increase in the number of microorganisms. Microorganisms 2021, 9, x FOR PEER REVIEW 13 of 17

Microorganisms 2021, 9, 1722 14 of 18 in the variants NPK (0.18 mg TPF/10 g), GAC3 (0.21 mg TPF/10 g), and Peat (0.29 mg TPF/10 g; Figure 7), which, however, corresponds to weak enzymatic activity.

FigureFigure 7. 7.The The change change of of soil soil dehydrogenase dehydrogenase activity activity with with various various methods methods of of bioremediation. bioremediation.

4. DiscussionMore high values of enzymatic activity at the beginning of the second growing seasonThe in results variants of the GAC3 correlation and Peat analysis correlate showed with a negative the rate correlation of the degradation between the of num- the berhydrocarbons of HOM and in the these content variants of TPH in winter. (r = 0.613–0.725 By the end for microfungiof the second and growingr = 0.626–0.730 season, forthe bacteria)enzyme andactivity a positive in all correlationthe variants, between except the fo numberr the variant of HOM BP, and slightly soil pH decreased, (r = 0.632–0.988 despite forthe microfungi increase in and the rnumber = 0.545–0.951 of microorganisms. for bacteria). At the same time, no correlation was found between the number of microorganisms and dehydrogenase activity, with the exception of4. theDiscussion variant BP when using HOM association, where a positive correlation was noted (r = 0.714–0.944).The results of the correlation analysis showed a negative correlation between the numberAt this of HOM stage ofand bioremediation, the content of theTPH biological (r = 0.613–0.725 system isfor unstable. microfungi Biochemical and r = 0.626– pro- cesses0.730 for in thebacteria) contaminated and a positive substrate correlation have lowbetween efficiency the number and multi-directionality. of HOM and soil pH In (r addition,= 0.632–0.988 the environmental for microfungi conditions and r = have 0.545–0.951 a great influence for bacteria). on the At number the same of HOM. time, The no research results show that, along with pH, moisture has a strong effect on biochemical correlation was found between the number of microorganisms and dehydrogenase processes in contaminated soils [51,52]. The activity of microorganisms decreases with low activity, with the exception of the variant BP when using HOM association, where a humidity, despite the temperature being close to the optimum, which is characteristic for positive correlation was noted (r = 0.714–0.944). soils with light granulometric composition and low organic content. At this stage of bioremediation, the biological system is unstable. Biochemical A number of studies demonstrate that both biostimulation and bioaugmentation processes in the contaminated substrate have low efficiency and multi-directionality. In increase the efficiency of soil cleansing processes from oil products. The choice of the addition, the environmental conditions have a great influence on the number of HOM. appropriate method depends on the environmental conditions [53]. The research results show that, along with pH, moisture has a strong effect on biochemical In our research the improvement of the water and air conditions (only loosening the processes in contaminated soils [51,52]. The activity of microorganisms decreases with top layer of soil) in the process of cleaning the contaminated areas was quite effective. low humidity, despite the temperature being close to the optimum, which is characteristic The decrease in the TPH content in the soil was 30% over the entire observation period. for soils with light granulometric composition and low organic content. The number of HOM and the activity of soil dehydrogenase did not change significantly, remainingA number at a very of studies low level, demonstrate which indicates that bo theth predominantly biostimulation physical and bioaugmentation and chemical degradationincrease the of efficiency the hydrocarbons. of soil cleansing processes from oil products. The choice of the appropriateBiostimulation method (i.e., depends using theon mineralthe environmental fertilizer together conditions with [53]. dolomite flour) increased the rateIn ofour hydrocarbons research the decay:improvement the TPH of content the water in the and soil air decreased conditions by (only an average loosening of 47% the intop 15 layer months. of soil) There in the was process an increase of cleaning in the the number contaminated of hydrocarbon-oxidizing areas was quite effective. microfungi The bydecrease 6–7 times, in the bacteria TPH content by 17 times, in the andsoil thewas activity 30% over of soilthe entire dehydrogenase observation by period. 2–6 times, The whichnumber indicates of HOM of biochemicaland the activity oxidation of soil of hydrocarbons.dehydrogenase did not change significantly, remainingBioaugmentation at a very low (use level, the which HOM indica association)tes the reducedpredominantly the TPH physical content and in chemical the soil bydegradation 45% in 15 of months, the hydrocarbons. which is comparable to the results obtained with biostimulation. A significant increase in the number of microorganisms occurs only after the repeated introduction of the microorganism associations after 1 year.

Microorganisms 2021, 9, 1722 15 of 18

The sorption-biological cleaning of the contaminated sites using the GAC also inten- siﬁed the biochemical oxidation of hydrocarbons (we noted an increase the number of HOM and the activity of dehydrogenase). It was possible to reduce the TPH content by 47–53%. However, the effectiveness of this method does not exceed the effectiveness of biostimulation but does increase the cost of cleaning. The positive effect of the introduction of sorbents into highly contaminated soils and grounds is due to the reversible sorption of toxic hydrocarbons and their metabolites. In biodegradation of n-alkanes, a primary alcohol is usually formed, followed by an aldehyde and a monocarboxylic acid. Further degradation of the carboxylic acid proceeds by oxidation with the formation shorter fatty acids, some of which are toxic and accumulate during hydrocarbon biodegradation. Iso- prenoid alkanes oxidate with the formation of dicarboxylic acids as the major degradative pathway. Cycloalkanes can be substrates for co-oxidation to form a ketone or alcohol. The bioremediation of aromatics involves the formation of a diol (followed by cleavage to a diacid) or a transdiol [54–56]. The sorbed hydrocarbons remain available to microorganisms. This is evidenced by an increase in the number of HOM, which occurs simultaneously with a decrease in TPH. A decrease in the toxic effect when using GAC was shown earlier in laboratory experiments [29]. The best results were obtained with the sorption-biological treatment of the peat. The content of hydrocarbons at the sites decreased by an average of 65%, which is 2.5 times more effective than in the variant A. In this variant, the maximum values of the HOM number and the activity of soil dehydrogenase were also noted. These patterns, noted by other researchers, are associated with the fact that sorbents based on GAC [57,58] and peat [59,60] provide both the sorption of petroleum hydrocarbons and are carriers of hydrocarbon-oxidizing microorganisms. However, peat may also contain various species of naturally occurring community that are capable of TPH degradation [61,62].

5. Conclusions Methods of biostimulation and sorption-biological treatment can be used for the bioremediation of contaminated sites in the subarctic zone of Russia, despite the difficult climatic conditions of the locations of the contaminated areas on the slopes of mountains and a high degree of soil degradation. Based on the results of the laboratory experiments, a scale of degrading activity of microfungi to petroleum hydrocarbons, was developed, which included three groups (with high, medium and low hydrocarbon oxidizing activity). Five species of microfungi that most intensively degraded petroleum hydrocarbons have been identified (Penicillium canescens st. 1, P. simplicissimum st. 1, P. commune, P. ochrochloron, P. restrictum). For soils and ground of light granulometric composition with a low content of organic matter but with a sufficiently high temperature regime, sorption-biological treatment using peat or GAC can be a more effective method of bioremediation. With this approach, the sorbent will not only bind hydrocarbons and their toxic metabolites but will also serve as a carrier for HOM and prevent the leaching of nutrients from the soil. The introduction of sorbents (mostly peat) increases the moisture capacity of the treated soil, which leads to an increase in the activity of HOM and the efficiency of bioremediation without the need for the periodic watering of the site. Good results were obtained due to the biostimulation of aboriginal hydrocarbon- oxidizing microbiota by the introduction of mineral fertilizers and liming. An increase in the number of microorganisms and dehydrogenase activity indicates the presence of a certain microbial potential in the soil and the possibility for the biochemical oxidation of hydrocarbons. The use of the HOM association, created based on bacteria and microfungi, which are active destructors of petroleum products, was less effective under these conditions and could be replaced by biostimulation techniques. Biostimulation techniques will reduce the cost of the treatment of the territory. The use of the considered bioremediation techniques contributes to the improvement of the ecological state of the contaminated area and to gradually recovering its biodiversity. In the future, we plan to undertake longer Microorganisms 2021, 9, 1722 16 of 18

observations of the model areas, as well as to evaluate the effectiveness of bioremediation methods (including the various carbon sorbents) and develop recommendations for the biological puriﬁcation of contaminated soils and coastal substrates from oil products in the Arctic and Subarctic.

Author Contributions: Conceptualization, V.A.M. and M.V.K.; methodology, V.A.M.; software, V.A.M.; validation, M.V.K., N.V.F. and G.K.V.; formal analysis, A.A.C.; investigation, V.A.M.; resources, A.A.C.; data curation, G.K.V.; writing—original draft preparation, A.A.C.; writing—review and editing, A.A.C.; visualization, V.A.M.; supervision, N.V.F.; project administration, M.V.K.; funding acquisition, M.V.K. and V.A.M. All authors have read and agreed to the published version of the manuscript. Funding: The field work has been supported by State scientific program AAAA-A18-118021490070-5. The chemical research has been supported by International Project Arctic Coast Bioremediation (KO 1001) within the framework of the Kolarctic CBC 2014–2020 Programme. The microbiological research has been supported by the RUDN University Strategic Academic Leadership Program. Acknowledgments: We acknowledge the head of the Pasvik nature reserve, Natalia Polikarpova, for help with the field research. Conflicts of Interest: The authors declare no conflict of interest. Disclaimer: This disclaimer informs readers that the views, thoughts, and opinions expressed in the text belong solely to the author.

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